ABSTRACT

We investigated the involvement of the CmeABC efflux pump in acquired resistance of Campylobacter jejuni to macrolides and tetracycline. Inactivation of the cmeB gene had no effect on macrolide resistance when all copies of the target gene carried an A2074C mutation. In contrast, the CmeABC pump significantly contributed to macrolide resistance when two or three copies of the 23S rRNA had an A2075G transition. Inactivation of the cmeB gene led to restoration of tetracycline susceptibility in the isolates examined. Complete susceptibility to tetracycline or macrolides, however, was not restored when phenylalanine-arginine β-naphthylamide was used. These data confirm contribution of the CmeABC efflux pump to acquired resistance of Campylobacter jejuni to tetracycline and macrolides.

Campylobacter species are the most common cause of bacterial human gastro-enteritis worldwide (1, 2). The most important Campylobacter species is Campylobacter jejuni, accounting for more than 90% of infections (12), with C. coli accounting for the remaining infections (18). Most cases of enteritis do not require treatment since they are of short duration, clinically mild, and self-limiting. Antimicrobial treatment is, however, necessary for systemic Campylobacter infections, infections in immunosuppressed patients, and in those with severe or long-lasting infections (1, 2). Erythromycin is still considered the drug of choice for treating Campylobacter gastroenteritis, although ciprofloxacin and tetracycline are used as alternative drugs (4, 18, 25).

To date, macrolide resistance in C. jejuni and C. coli has mainly been attributed to mutations in domain V of the 23S rRNA target gene at positions 2074 and 2075, which correspond to positions 2058 and 2059 in Escherichia coli (10, 19, 27, 28). Tetracycline resistance (Tcr) is primarily mediated by a plasmid-encoded tet(O) gene (11, 26). Tet(O), a ribosomal protection protein, confers resistance by displacing tetracycline from its primary binding site on the ribosome (26).

Recently, a Campylobacter multidrug efflux pump (named CmeABC) contributing to antimicrobial resistance was characterized and found to be widely distributed in different isolates of C. jejuni (6, 14, 15). This efflux pump belongs to the resistance nodulation division (RND) family of transporters and is encoded by an operon of three genes located on the chromosome (14). The CmeABC efflux pump consists of three components: a periplasmic fusion protein, CmeA; an inner membrane drug transporter, CmeB; and an outer membrane protein, CmeC (14). Constitutive expression of the cmeABC operon occurs at a moderate level in wild-type Campylobacter strains (13), and the CmeABC efflux pump extrudes a wide variety of compounds such as dyes, detergents, and antimicrobial agents of various families (14, 23).

Previous studies showed that active efflux contributes to the intrinsic and low-level macrolide resistance mostly in C. coli (6, 9, 17, 20, 21). On the other hand, a very limited number of studies investigated the contribution of active efflux to high-level macrolide resistance in C. jejuni isolates (8, 17). The extent of the contribution of the CmeABC efflux system to the development of high-level macrolide resistance in Campylobacter is still controversial. It is possible that the contribution of the CmeABC efflux system is affected by the number of mutated copies of the 23S rRNA gene or the site of the mutation in the target gene. The interplay between ribosomal mutations and active efflux in the development of high-level macrolide resistance in C. jejuni and C. coli requires further evaluation. More information regarding the role of the CmeABC efflux pump in the emergence of high-level tetracycline-resistant isolates of C. jejuni and C. coli is also required to advance our understanding of the significance of active efflux in the development of multidrug resistance phenotype in Campylobacter.

The goal of the present study was to investigate the contribution of the CmeABC efflux pump to macrolide and tetracycline resistance in C. jejuni. In addition, the efflux pump inhibitor, PAβN, was also shown to inhibit both macrolide and tetracycline resistance but its inhibitory effect was less than that noted in the cmeB::Kanr mutants.

MATERIALS AND METHODS

Campylobacter isolates and culture conditions.Various C. jejuni isolates involved in the present study are listed in Table 1. Campylobacter isolates were routinely grown in Mueller-Hinton broth or agar at 37°C under microaerobic conditions. The bacteria were stored at −70°C in brain heart infusion broth supplemented with 25% glycerol.

Determination of MICs.Determination of the MICs of erythromycin, clarithromycin, and tetracycline was performed by using the agar dilution method recommended by the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) (7). To ensure reproducibility, MIC determinations were repeated at least twice. C. jejuni strain ATCC 33560 was used as the quality control organism (Table 1). Isolates were considered resistant to erythromycin, clarithromycin, and tetracycline if they had MICs of ≥8, ≥8, and ≥16 μg/ml, respectively. To examine the effect of PAβN on the active efflux of macrolides and tetracycline, the MICs of erythromycin, clarithromycin, and tetracycline were determined by using E-test strips (AB Biodisk, Solna, Sweden) in the presence or absence of the efflux pump inhibitor, as previously described (10). PAβN was incorporated into Mueller-Hinton agar plates at 10, 20, 30, or 40 μg/ml. This experiment was repeated at least twice to ensure reproducibility.

Construction of the efflux mutants of C. jejuni.The cmeB::Kanr mutants of C. jejuni isolates were constructed by natural transformation with genomic DNA (0.5 to 1 μg) of a 81-176 cmeB::Kanr mutant as described previously (14, 29). The resulting mutants were confirmed by PCR using the primers cmeB-F (5′-GATGGCTCAATGAGTGCAGTAG-3′) and cmeB-R (5′-CATCTACAACGATCCCTATGGC-3′) that flank the inserted kanamycin resistance cassette. The size of the PCR amplicon in the case of the efflux mutants was about 2.2 kb compared to about 800 bp for the corresponding parent isolates indicating the insertion of a 1.4-kb kanamycin resistance cassette. PCR amplification was carried out in 50-μl reaction volumes containing oligonucleotide primers at 0.5 μM each; dATP, dCTP, dGTP, and dTTP at 200 μM each; 1× reaction buffer (50 μM KCl, 10 mM Tris-Hcl [pH 8.3], 2 mM MgCl2, 0.01% [wt/vol] gelatin); 1 U of Accuprime (Invitrogen); and the genomic DNA template (100 ng). The PCR conditions were as follows: 95°C for 1 min, 55°C for 1 min, and 68°C for 1 min, repeated for 30 cycles, followed by a 10-min final extension at 68°C. In order to confirm that the cmeB::Kanr mutants of 15-21, 23-51, 25-19, and 25-45 still carry the tet(O) gene, PCR amplification was carried out to amplify about 500 bp of the tet(O) gene using the PCR primers and amplification conditions described previously (11). All PCR amplicons were visualized by using agarose gel electrophoresis according to a standard method (24).

Transfer of the tet(O) gene into efflux mutants of E. coli.Plasmid pDOB47 was isolated from E. coli DH5α, DMT47 (11), by using a QIAGEN Mini-Plasmid kit (QIAGEN, Mississauga, Ontario, Canada). Plasmid pDOB47 was transferred to EW1b, a tolC efflux mutant of E. coli (30), which had been made competent by calcium chloride treatment (24). The transformants were selected on LB plates containing 50 μg of kanamycin/ml. A number of the resulting transformants were screened to ensure the transfer of pDOB47 plasmid. The tetracycline MIC was determined in eight of the tolC transformants by using the agar dilution method as described above. In parallel, E. coli DMT47 and EW1b were included during MIC determinations as positive and negative controls, respectively. The MIC determination was repeated at least twice to ensure reproducibility.

RESULTS AND DISCUSSION

Role of the CmeABC efflux pump in high-level resistance to macrolides in C. jejuni.Previous studies on the role of the active efflux in conferring antimicrobial resistance in C. jejuni and C. coli used either insertional inactivation of the cmeB gene to inactivate the RND CmeABC system (5, 6, 9) or inhibition of the general active efflux through the use of the inhibitor, PAβN (8, 16, 17, 20). A few studies examined the role of the CmeABC efflux pump in conferring high-level resistance to macrolides in isolates of C. jejuni and C. coli that mainly carried an A2075G transition in the three copies of the 23S rRNA gene (5, 6, 8).

We wanted to assess the contribution of the CmeABC pump to high-level macrolide resistance in an isolate that had an A2074C transversion. In addition, we explored the extent of the efflux pump contribution in the case of isolates carrying an A2075G transition in different numbers of copies of the 23S rRNA gene. We also felt it would be valuable to compare the efficiency of the two approaches commonly used to assess the role of the CmeABC pump in the development of antimicrobial resistance in C. jejuni and C. coli.

In the present study, three macrolide-resistant isolates of C. jejuni—UA709, 001B-22, and 001B-40 (Table 1)—were investigated. UA709 carried an A2074C transversion in the three copies of the 23S rRNA gene. Isolate 001B-22 had an A2075G transition in only two copies of the 23S rRNA gene, whereas isolate 001B-40 carried the same mutation in all copies of the target gene. Insertional inactivation of the cmeB gene in isolate UA709 had no effect on the MIC of erythromycin or clarithromycin (Table 2). However, 001B-22 cmeB::Kanr mutants showed 64- and 16-fold decreases in the MICs of erythromycin and clarithromycin, respectively, resulting in complete restoration of susceptibility (Table 2). The 001B-40 cmeB::Kanr mutants exhibited >128- and >32-fold decreases in the MICs of erythromycin and clarithromycin, respectively (Table 2); however, the mutants remained resistant to erythromycin and clarithromycin but with a low-level resistance phenotype (Table 2).

Effect of inactivation of the CmeABC efflux pump in C. jejuni on resistance to erythromycin and clarithromycin

The MICs of erythromycin and clarithromycin were also determined for isolates UA709 and 001B-22 in the presence or absence of the efflux pump inhibitor, PAβN. Under these conditions, comparison of the bacterial growth of UA709 and 001B-22 in the absence or presence of 20 and 30 μg of PAβN/ml, respectively, did not show any partial inhibition of the growth of the isolates examined. Again, the resistance level of isolate UA709 was not affected by the presence of PAβN (Table 2). Whereas the inhibition of the active efflux by PAβN in the case of 001B-22 slightly reduced the resistance level to erythromycin (∼3-fold decrease) and clarithromycin (∼2-fold decrease). A similar observation has been reported by Payot et al. (20), who found that the erythromycin resistance of an isolate that carried the A2075G mutation in two copies of the target gene was very slightly altered due to the inhibition of the active efflux by PAβN. It is worth mentioning that several investigators have reported that the use of 40 mg of PAβN/liter inhibited the growth of many high-level resistant Campylobacter strains (10, 17). This suggests that the effect of PAβN on the growth of Campylobacter could be isolate dependent, necessitating careful optimization of the inhibitor concentration, when used in similar investigations.

The results reported here suggest that the active efflux mediated by the CmeABC pump has no contribution to the high-level resistance to macrolides in isolates that carry an A2074C mutation in the three copies of the 23S rRNA gene. To our knowledge, this is the first report describing the effect of insertional inactivation of the CmeABC pump on macrolide resistance in C. jejuni carrying an A2074C transversion. It is tempting to speculate that isolate UA709 may carry another, as-yet-uncharacterized, efflux pump or resistance mechanism that could act synergistically with the target modification in mediating high-level resistance to macrolides, a mechanism that was not reduced by knocking out the CmeABC pump. Our results also indicate that the CmeABC pump significantly contributes to the development of high-level macrolide resistance when two or three copies of the 23S rRNA gene carried an A2075G mutation. This was in agreement with what has previously been reported by Cagliero et al. (6).

The data presented here strongly suggest that the use of PAβN is not the ideal approach for the assessment of the role of the active efflux in conferring antimicrobial resistance in Campylobacter spp. compared to the insertional inactivation approach. The limited effect of PAβN on the CmeABC pump reported here could be due to an inability to use a concentration of the inhibitor higher than 30 μg/ml, since attempts above this concentration were found to have an inhibitory effect on the growth of the isolates examined. A similar observation has previously been reported by Cagliero et al. (6), who found that the use of PAβN only led to a 4- to 8-fold reduction in erythromycin MICs of high-level resistant isolates of C. coli, whereas, the MICs greatly decreased (128- to 512-fold) upon cmeB gene inactivation (6).

Contribution of CmeABC efflux pump to high-level resistance to tetracycline in C. jejuni.The possible interplay between the ribosomal protection, mediated by the tet(O) gene, and the active efflux mediated by the CmeABC pump in conferring tetracycline resistance (Tcr) was examined in four isolates of C. jejuni (Table 1). These isolates were found to carry the tet(O) gene as a resistance marker as shown by the PCR amplification of about 500 bp of the tet(O) gene. In addition, these isolates exhibited variable levels of Tcr (Table 3). C. jejuni isolate 81-176 [tet(O)+] was also included as a positive control (Table 3). The assessment of the role of the CmeABC efflux pump in acquired Tcr was analyzed by using the two approaches described above.

Effect of inactivation of the CmeABC efflux pump in C. jejuni on resistance to tetracycline

The existence of the tet(O) gene in the resulting cmeB::Kanr mutants was confirmed by PCR amplification as described above. Inactivation of the cmeB gene in the resistant isolates examined led to a 16- to 128-fold decrease in tetracycline MICs, resulting in the complete restoration of tetracycline susceptibility (Table 3). This indicates that tet(O)-mediated Tcr in case of C. jejuni is completely abolished when the active efflux in C. jejuni is inactive. This is probably due to the cell being flooded with tetracycline, completely overwhelming the ribosomal protection provided by the Tet(O) protein. This highlights the major contribution of the CmeABC efflux pump to the acquired Tcr in C. jejuni.

On the other hand, inhibition of the CmeABC efflux pump by the addition of 30 μg of PAβN/ml did not lead to the same effect (Table 3). Tetracycline MICs in all isolates investigated were slightly reduced in the presence of PAβN, but tetracycline susceptibility was not restored (Table 3). This might indicate that PAβN is poorly effective in competing with tetracycline in the case of C. jejuni. It is possible that the limited effect of PAβN on Tcr levels of the isolates involved in the present study was due to the inability to increase the concentration of PAβN more than 30 μg/ml. Our results again show that the insertional inactivation of the CmeABC efflux pump in C. jejuni is the most efficient tool in the assessment of the contribution of the active efflux to antimicrobial resistance.

The AcrAB-TolC efflux pump in E. coli is a homologue of the CmeABC pump in Campylobacter, and both pumps belong to the RND family of the efflux pumps (22). To confirm the significant contribution of active efflux (mediated by another efflux pump belonging to the RND family) in Tcr, the plasmid pDOB47, which has the tet(O) gene with approximately 1 kb upstream region cloned into pRY107 (11), was introduced into the tolC5 deletion mutant (EW1b) of E. coli by transformation. In parallel, pDOB47 was also transferred to the wild-type E. coli DH5α. Tetracycline MICs were compared for the transformants of the wild-type E. coli, DH5α, and those of the tolC5 mutants using the agar dilution method. It was found that there was a fourfold decrease in tetracycline MIC of the transformants of the tolC5 mutants (16 μg/ml) compared to the transformants of the wild-type strain (64 μg/ml). This confirms the existence of a synergistic effect between the active efflux, mediated by AcrAB-TolC efflux pump, and the Tet(O) protein. This suggests that the cooperation, observed in C. jejuni isolates examined in the present study, between ribosomal protection and active efflux in conferring high-level Tcr could be operational in E. coli. The slight difference between the tetracycline MIC of the transformants of the tolC5 mutants (16 μg/ml) and the corresponding MIC in the case of the efflux mutants of C. jejuni investigated (4 to 8 μg/ml) might be attributed to the high copy number of pDOB47 plasmid transferred to the tolC5 mutant.

We propose that high-level resistance to tetracycline in C. jejuni, conferred by Tet(O), actually results from its cooperation with CmeABC, which would capture tetracycline, released from the ribosome by the action of Tet(O) protein, and then extrude the drug into the external medium.

Evidence of the presence of another PAβN-sensitive efflux pump in C. jejuni.To examine whether or not the efflux mutants investigated had an additional efflux system active against macrolides and tetracycline, the effect of the efflux pump inhibitor, PAβN, on the efflux mutants examined was evaluated. With the exception of UA709 efflux mutant, which was not affected by the addition of PAβN, all other mutants examined displayed smaller magnitudes of reduction (fourfold) in the MICs of macrolides and tetracycline. This suggests that an additional efflux mechanism, distinct from CmeABC, could be active in Campylobacter, although it has a minor contribution to macrolide and tetracycline resistance. These results were in agreement with a previous observation by Mamelli et al. (17).

ACKNOWLEDGMENTS

We thank Q. Zhang for providing the cmeB::Kanr mutant of 81-176.

This study was supported by the Canadian Institute of Health Research and the Natural Science and Engineering Research Council. D.E.T. is a senior investigator with the Alberta Heritage Foundation for Medical Research.